Abstract

Brain-selective kinases (BRSK1 and BRSK2) are serine/threonine kinase membersof the AMPK-related family of protein kinases, the majority of which are regulatedby the upstream kinase LKB1 whilst AMPK itself is regulated by CaMKK. TheBRSKs are highly expressed in brain and have been implicated in neuronalpolarization and the regulation of neurotransmitter release. They have also beenshown to be involved in the basal phosphorylation of tau at the Alzheimer‟s disease(AD) related residue, serine 262, and are highly expressed in areas of the brainaffected by AD, namely the hippocampus and the cortex. I have utilised the modelorganism Drosophila melanogaster to investigate interactions between transgenicallyexpressed human tau, human BRSKs and upstream regulatorsSelective over-expression of 0N4R human tau in the Drosophila eye resulted in adisruption of eye morphology. In contrast, over-expression of human wild typeBRSK2 (B-WT) had no obvious effect on the eye. However, co-expression of bothtau and B-WT resulted in a neurodegenerative phenotype more severe than tau alone.This enhancement of phenotype was not observed when BRSK2 was expressed thateither lacked the activating phosphorylation site (non-phosphorylatable, B-NP) orthat is unable to bind ATP (kinase dead, B-KD). Co-expression of human tau and BWTsignificantly elevated tau phosphorylation at S262, suggesting that S262 is a keyresidue for tau-induced toxic phenotypes and the BRSK/tau interaction I observe. Insupport of this, no phenotype was observed in flies expressing the S262A variant ofhuman tau with or without B-WT.To establish the upstream kinases responsible for activating human BRSK2 inDrosophila I removed endogenous Drosophila LKB1 by RNAi. This prevented theenhanced degeneration of the eye caused by tau/B-WT co-expression, demonstratingthat LKB1 is a key upstream regulator of BRSK2. I also found that down regulationof the Drosophila CaMKK homologue, CG17698, by the same method, amelioratedB-WT induced eye degeneration implicating a calcium-dependent pathway in theregulation of BRSK. Over-expression of human CaMKKα in the CG17698 RNAibackground prevented the rescue seen with CG17698 RNAi. Over-expression ofcac1, a calcium channel subunit, in the presence of B-WT and human tauexacerbated the B-WT induced eye phenotype in a B-WT dependent manner,supporting the hypothesis that the human tau and B-WT interaction can be regulatedin a calcium-dependent manner.Expression of total BRSK2, LKB1 and CaMKK were not altered in human postmortemAD brain tissue when compared to control. However, with the exception ofLKB1, due to limited reagents and time constraints I was unable to investigate theproportion of phosphorylated (and thus active) to total kinase.This study defines a novel Ca2+ -dependent regulatory pathway to tau, which maycontribute to AD and other tauopathies.